FIG S1 Examination of eif4b expression after virus infection. (A) A549 cells

Transcription

1 Supplementary Figure Legends FIG S1 Examination of expression after virus infection. () 549 cells were infected with herpes simplex virus (HSV) (MOI = 1), and harvested at the indicated times, followed by Western blotting with indicated antibodies. () 549 cells were infected with HSV as described in () and followed by RT-PCR to detect mrn levels. (C) MEFs were uninfected or infected with WSN viruses for 12 h, and mrn levels were analyzed by RT-PCR. (D, E) Mice intranasally infected with or without WSN for three days were sacrificed, and the mrn levels of in mouse lung (D), thymus, spleen and liver (E) were detected by RT-PCR. FIG S2 shrn-based knockdown of host RIG-I, TLR3, MD5, and viral NS1. (,, C) shrn-based knockdown of RIG-I (), TLR3 () and MD5 (C) was analyzed by RT-PCR to determine the interference efficiency. served as an internal control. (D) Shown are 549 cell lines stably expressing shrns targeting either luciferase (control) or NS cells were infected with pseudolentivirus particles produced in 293T cells co-transfected with psih-h1-gfp containing the target sequences and plasmids PLP1, PLP2 and pcl-vsvg. The cells were sorted by GFP expression with flow cytometry. FIG S3 lteration of expression significantly affects influenza virus replication. () 549 cells stably expressing the shrns targeting or luciferase were infected with WSN viruses (MOI =.1). The supernatants of cell 1

2 culture were examined for the viral titers by plaque assay (p.i. = 22 h). () 549 cells overexpressing and control cells were infected with WSN viruses as described in (). Viral titers in the supernatants of these cells were examined by plaque assay (p.i. = 22 h). P <.1 (Student s t test). FIG S4 Representative lung and thymus images showing pulmonary lesions and thymic atrophy after IV infection. Wild-type () and knockdown transgenic () mice were mock infected or infected intranasally with WSN for 3 days. Then mice were sacrificed, and the lungs and thymuses were collected. () and () are representative images from three independent experiments. FIG S5 Examination of the expression of ISGs, PKR and p58ipk. () The differentially expressed genes in 549 cells infected with or without WSN influenza virus were analyzed by cdn microarray in our previous study ( access number GSE32878). Shown are representative genes whose expressions were significantly changed. () 549 cells expressing shrns targeting or luciferase (control) were infected with or without WSN for 12 h, and then the mrn levels of Mx, ISG15, IFITM1, IFITM2 and were analyzed by RT-PCR. (C) -knockdown 549 cells and the control cells were uninfected or infected with WSN viruses for 12 h. The cells were harvested and cell extracts were prepared for Western blotting using indicated antibodies. The results are representative of three independent experiments. (D, E) shrn based 2

3 knockdown of (D) and ISG15 (E) under the condition of IFNβ treatment was analyzed by Western blotting using the antibodies as indicated. FIG S6 Effects of altering levels on the expression of and ISG15. () Western blot analysis of the lysates of knockdown 549 cells and the control cells treated with or without IFNβ (5 U/ml) for 12 h. () 293T cells were co-transfected with (.5 μg) and either Empty Vector (2.5 μg), Flag- () (2.5 μg), or Flag- (S46/S422) (2.5 μg) for 32 h. The cells were then harvested and analyzed by Western blotting using indicated antibodies. This result is representative of three identical experiments. (C) protein levels under the condition of WSN infection in Fig. 7G were quantitated by densitometry, and normalized to levels. In each experiment, level in MEFs is 1. (D) Western blotting was performed as described in the figure legend for Fig. 7G. More samples were loaded and a longer exposure time was used. (E) protein levels under the condition of WSN infection in Fig. 7H were quantitated by densitometry, and normalized to levels. In each experiment, level in the spleen of mice is 1. (F) Wild-type and knockdown transgenic mice intranasally infected with or without WSN viruses for 3 d were sacrificed, and the lungs were homogenized, followed by Western blotting using indicated antibodies. (G) protein levels under the condition of WSN infection in (F) were quantitated by densitometry, and normalized to levels. In each experiment, level in the lung of mice is 1. P <.1 (Student s t test). 3

4 Supplementary Figure S1 C HSV (h) HSV (h) HSV-gC WSN NP MEFs - + D WSN NP Lung - + E Thymus Spleen Liver WSN - +

5 Supplementary Figure S2 D sh-rig-i WSN- WSN+ F GFP C RIG-I sh-tlr3 TLR3 sh-md5 MD5 WSN- WSN- WSN+ WSN+ sh-ns1-1 sh-ns1-2

6 Supplementary Figure S3 Virus titer (1 5 PFU/ml) sh- Luciferase sh Virus titer (1 5 PFU/ml) Control pnl-

7 Supplementary Figure S4 Lung WSN(-) WSN(+) Thymus Lung WSN(-) WSN(+) Thymus

8 Supplementary Figure S5 C WSN- WSN+ WSN- WSN+ sh- PKR p58ipk MX1 ISG15 IFITM1 IFITM2 TLCD1 FEN1 OGDH PLK1 CPLX2 Up regulation 1-1 Down regulation D sh- IFNβ sh- Mx ISG15 IFITM1 IFITM2 E WSN- WSN+ sh-isg15 ISG15 IFNβ

9 Supplementary Figure S6 sh- IFNβ- IFNβ+ Empty Vector () (S46/S422) ISG15 Flag- C protein levels E protein levels G D NP F WSN- WSN WSN- WSN+ protein levels